The matrix metalloproteinases (MMPs) are zinc metalloproteinases that degrade components of the extracellular matrix. They play major roles in diseases including arthritis, cancer and atherosclerosis. The activities of MMPs are regulated by endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs)-1, to -4. Our long-range goals are to understand how TIMPs inhibit MMPs and to use this information to engineer variant TIMPs that selectively inhibit individual or chosen groups of metalloproteinases. These targeted TIMPs will be tested for efficacy in alleviating the progression of diseases associated with increased degradation of the extracellular matrix. Our recent finding that TIMP-3 is a potent inhibitor of two aggrecanases (ADAMTS-4 and ADAMTS-5), key enzymes in the degradation of the cartilage proteoglycan, aggrecan, leads us to further investigate and test TIMP-3, and other TIMP variants, as inhibitors for preventing the progression of arthritis. To achieve these goals we will elucidate the structural basis of TIMP specificity for aggrecanases and MMPs and engineer TIMPs that are targeted for metalloproteinases involved in cartilage degradation. These inhibitors will be tested for effectiveness in ex vivo and in vivo models of rheumatoid arthritis (RA) and osteoarthritis (OA). The Specific Aims are: (1) to investigate the mechanism of inhibition of aggrecanases by TIMP-3 and generate specific inhibitors of these enzymes; (2) to produce TIMP variants that selectively inhibit collagenases (MMP-1 and -13), gelatinase A (MMP-2), or MT1-MMP; (3) identify and characterize low-molecular weight peptide inhibitors employing non-toxic variants of sarafotoxin, an analogue of the unique inhibitory region of TIMPs; (4) characterize the structural and physical basis of strong and specific metalloproteinase binding in TIMPs; (5) test recombinant TIMP-3 and other wild-type and variant TIMPs for their ability to prevent cartilage breakdown using the cartilage explant system; (6) test the efficacy of TIMP-3 and TIMP variants as potential blockers of cartilage degradation in the collagen-induced arthritis model of RA, and in the STR/ort mouse OA model; and (7) identify metalloproteases that act in articular cartilage breakdown during the progression of OA in humans. These studies will produce mechanistic and structural information about the interactions of TIMPs and metalloproteinases and new insights into therapeutic approaches for arthritis.